Targeting microRNAs in the tumor microenvironment with pHLIP conjugated next generation chemically modified PNAs

弥漫性大B细胞淋巴瘤(DLBCL)是美国一种常见的癌症，导致数万人死亡 每年，它的发病率都在上升。MicroRNAs(MiRNAs)是一种小的非编码RNA，经常被误认为是 在淋巴瘤和白血病中受到调节。尤其是miR-155在多发性淋巴瘤/白血病亚细胞中表达上调。 类型(特别是DLBCL)。此外，有直接证据表明，它的过度表达会导致人类DLBCL DLBCL异种移植和转基因小鼠模型。有趣的是，由于miR-1过度表达而导致的肿瘤 155依靠miRNA的持续表达生存，因为如果miRNA被撤回，肿瘤 通过细胞凋亡快速退化。这种“癌基因成瘾”的特性使这类miRNAs成为理想的靶标。 用于未来的抗癌治疗，并推动了通过肿瘤内抑制oncomiR-155的临床试验的启动 皮肤T细胞淋巴瘤(CTCL)和DLBCL中的钴胺递送。在取得显著进展的同时， 在过去的几年中，随着FDA批准反义和RNAi药物，治疗性miRNA靶向 仍然落后，主要是因为与选择性递送和毒性有关的问题。 在这里，我们建议利用癌基因成瘾的惊人特性来开发潜在的量身定做的治疗方法 以安全有效的方式对抗这一关键的元凶。我们之前发布了一种方法，以 用PHLIP(低pH值)靶向酸性肿瘤微环境的抗miR-155肽核酸 插入多肽)，对淋巴瘤小鼠模型有显著效果。Phlip是一种由36个氨基酸组成的肽 在低pH(pH 6.5)下采用α-螺旋构象变化，促进其C末端插入 酸性肿瘤微环境中的脂质双层。在这里，我们提出了一个多学科项目，重点是 翻译下一代PNA和PHLIP技术，以实现高度特定的靶向和更高的效率 在miR-155成瘾癌症的酸性组织中靶向致癌miR-155，具有有利的治疗窗口。 与大多数核酸不同，PNA是一种人工合成的DNA模拟物，其中磷酸二酯的骨架是 被中性N-(2-氨基乙基)甘氨酸主链取代。PNAs可以与高密度的单链靶标结合 特异性和亲和力强，不受蛋白酶或核酸酶的影响，使PNA成为理想的 靶向miRNA。为了进一步提高反干扰PNA的有效性，我们将开发一类新的PNA 类似物，被指定为伽马PNA(GPNA)的构象预先组织的，因此具有优势 结合以及溶解性能，应增加其作为抗干扰剂的有效性。作为证明 我们稳定的下一代PHLIP偶联、化学修饰的伽马PNA(GPNAs)作为癌症的原理 治疗，我们建议在这个项目中测试淋巴瘤癌症模型中的传递和疗效(细胞系， 异种移植、PDX和GEMM)，最终目标是制备一种适合临床试验的药物。调查 在这些利用针对酸性微环境的新型低聚物来开发miRNA成瘾的方法中，将 为针对淋巴瘤/白血病和其他肿瘤的分子靶向癌症疗法的发展提供信息。

Diffuse large B-cell lymphoma (DLBCL) is a common cancer in the US, causing tens of thousands of deaths each year and its incidence is on the rise. MicroRNAs (miRNAs) are small non-coding RNAs that are often mis- regulated in lymphoma and leukemia. In particular, miR-155 is up-regulated in multiple lymphoma/leukemia sub- types (notably DLBCL). Plus, direct evidence exists to show that its over-expression causes DLBCL in human DLBCL xenografts and transgenic mouse models. Interestingly, the tumors induced by over-expression of miR- 155 depend on the continued expression of the miRNA for survival, since if the miRNA is withdrawn, the tumors rapidly regress via apoptosis. This property of “oncogene addiction” makes this class of miRNAs ideal targets for future anti-cancer therapy and promoted the initiation of a clinical trial to inhibit oncomiR-155 via intratumoral delivery of Cobomarsen in Cutaneous T-cell Lymphoma (CTCL) and DLBCL. While remarkable progress has been made in the past few years with FDA approvals of antisense and RNAi drugs, therapeutic miRNA targeting still lags behind, largely due to issues related to selective delivery and toxicity. Here we propose to exploit the striking nature of oncogene addiction to develop potential therapeutics tailored to antagonize this crucial oncomiR in a safe and effective manner. We previously published an approach to target anti-miR-155 peptide nucleic acids (PNAs) to the acidic tumor microenvironment using pHLIP (pH Low Insertion Peptide), with significant effects in a mouse model of lymphoma. pHLIP is a 36-amino acid peptide that adopts an α-helical conformational change at low pH (< pH 6.5) that facilitates insertion of its C-terminus across lipid bilayers in the acidic tumor microenvironment. Here we propose a multi-disciplinary project focused on translating next-generation PNA and pHLIP technology to allow highly-specific targeting and improved efficacy targeting oncogenic miR-155 in acidic tissues in miR-155-addicted cancers, with a favorable therapeutic window. Unlike most nucleic acids, PNAs are a synthetic DNA mimics in which the phosphodiester backbone is substituted with a neutral N-(2-aminoethyl) glycine backbone. PNAs can bind single-strand targets with high specificity and affinity and are not susceptible to proteases or nucleases, making PNAs ideal molecules for targeting miRNAs. To further improve the effectiveness of antimiR PNAs, we will exploit a new class of PNA analogs, designated gamma PNAs (gPNAs) that are conformationally pre-organized and so have advantageous binding as well as solubility properties that should increase their effectiveness as antimiR agents. As proof of principle for our stable next generation pHLIP-conjugated, chemically modified gamma PNAs (gPNAs) as cancer therapeutics, we propose to test delivery and efficacy in lymphoma cancer models in this project (cell lines, xenografts, PDX and GEMMs), with the ultimate goal of preparing a drug suitable for clinical trials. Investigation of these methods to exploit miRNA addiction with novel oligomers targeted to the acidic microenvironment will inform the development of molecularly targeted cancer therapeutics for lymphoma/leukemia and other tumors.